2002/225/EC: Commission Decision of 15 March 2002 laying down detailed rules for the implementation of Council Directive 91/492/EEC as regards the maximum levels and the methods of analysis of certain marine biotoxins in bivalve molluscs, echinoderms, tunicates and marine gastropods (Text with EEA relevance) (notified under document number C(2002) 1001)
Date: 2002-03-15
Institutions: European Commission
Subjects: European standard, dangerous substance, health control, mollusc

L 75/62

EN

Official Journal of the European Communities

16.3.2002

COMMISSION DECISION

of 15 March 2002

laying down detailed rules for the implementation of Council Directive 91/492/EEC as regards the
maximum levels and the methods of analysis of certain marine biotoxins in bivalve molluscs,
echinoderms, tunicates and marine gastropods

(notified under document number C(2002) 1001)

(Text with EEA relevance)

(2002/225/EC)

THE COMMISSION OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European
Community,

Having regard to Council Directive 91/492/EEC of 15 July
1991 laying down the health conditions for the production
and the placing on the market of live bivalve molluscs (1), as
last amended by Directive 97/79/EC (2), and in particular
Chapter V, paragraphs 3 and 5, of the Annex thereto,

Whereas:

(6)

(7)

The proposed maximum levels are based on provisional
data and should be re-evaluated when new scientific
evidence becomes available.

The measures provided for in this Decision are in
accordance with the opinion of the Standing Veterinary
Committee,

HAS ADOPTED THIS DECISION:

Article 1

(1)

(2)

(3)

(4)

(5)

Chapter V, point 7, of
the Annex to Directive 91/
492/EEC provides that the customary biological testing
methods must not give a positive result to the presence
of diarrhetic shellfish poisoning (DSP) in the edible parts
of molluscs (the whole body or any part edible sepa-
rately).

such as

those of

It has been scientifically proven that certain marine
biotoxins
the diarrhetic shellfish
poisoning (DSP) complex (okadaic acid (OA) and dino-
physistoxins (DTXs)) and also yessotoxins (YTXs), pecte-
notoxins (PTXs) and azaspiracids (AZAs), pose a serious
hazard to human health when present above certain
limits in bivalve molluscs, echinoderms,
tunicates or
marine gastropods.

In the light of recent scientific studies it is now possible
to establish maximum levels and methods of analysis for
those biotoxins.

Maximum levels and methods of analysis should be
harmonised and be implemented by the Member States
in order to protect human health.

In addition to biological
testing methods, alternative
detection methods such as chemical methods and in vitro
assays should be accepted if it is demonstrated that the
performance of the chosen methods is not less effective
than the performance of the biological method and that
their implementation provides an equivalent
level of
public health protection.

(1) OJ L 268, 24.9.1991, p. 1.
(2) OJ L 24, 30.1.1998, p. 31.

This Decision lays down the maximum levels for the marine
biotoxins of the diarrhetic shellfish poisoning (DSP) complex
(okadaic acid and dinophysistoxins), yessotoxins, pectenotoxins
and azaspiracids and the methods of analysis to be used for
their detection. It applies to bivalve molluscs, echinoderms,
are
tunicates
intended for
further processing
immediate human consumption or
before consumption.

and marine gastropods

that
for

Article 2

The maximum level of okadaic acid, dinophysistoxins and
pectenotoxins together in the animals referred to in Article 1
(the whole body or any part edible separately) shall be 160 µg
of okadaic acid equivalents/kg. The methods of analysis are set
out in the Annex.

Article 3

The maximum level of yessotoxins in the animals referred to in
Article 1 (the whole body or any part edible separately) shall be
1 mg of yessotoxin equivalent/kg. The methods of analysis are
set out in the Annex.

Article 4

The maximum level of Azaspiracids in the animals referred to
in Article 1 (the whole body or any part edible separately) shall
be 160 µg of azaspiracid equivalents/kg. The methods of
analysis are set out in the Annex.

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When the results of the analyses performed demonstrate discrepancies between the different methods, the
mouse bioassay should be considered as the reference method.

Article 5

This Decision is addressed to the Member States.

Article 6

Done at Brussels, 15 March 2002.

For the Commission

David BYRNE

Member of the Commission

L 75/64

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Official Journal of the European Communities

16.3.2002

ANNEX

Detection methods

Biological methods

A series of mouse bioassay procedures, differing in the test portion (hepatopancreas or whole body) and in the solvents
used for the extraction and purification steps, can be used for detection of the toxins mentioned in Article 1. Sensitivity
and selectivity depend on the choice of the solvents used for the extraction and purification steps and this should be taken
into account when making a decision on the method to be used,

in order to cover the full range of toxins.

A single mouse bioassay involving acetone extraction can be used to detect okadaic acid, dinophysistoxins, pectenotoxins
and yessotoxins. This assay may be complemented if necessary with liquid/liquid partition steps with ethyl acetate/water
or dichloromethane/water to remove potential interferences. Azaspiracids detection at the regulatory levels by means of
this procedure requires the use of the whole body as the test portion.

Three mice should be used for each test. The death of two out of three mice within 24 hours after inoculation into each
of them of an extract equivalent to 5 g of hepatopancreas or 25 g whole body should be considered as a positive result
for the presence of one or more of the toxins mentioned in Article 1 at levels above those established in Article 2, 3 and
4.

A mouse bioassay with acetone extraction followed by liquid/liquid partition with diethylether can be used to detect
okadaic acid, dinophysistoxins, pectenotoxins and azaspiracids but it cannot be used to detect yessotoxins as losses of
these toxins may take place during the partition step. Three mice should be used for each test. The death of two out of
three mice within 24 hours after inoculation into each of them of an extract equivalent to 5 g of hepatopancreas or 25 g
whole body should be considered as a positive result for the presence of okadaic acid, dinophysistoxins, pectenotoxins
and azaspiracids at levels above those established in Article 2 and 4.

The rat bioassay can detect okadaic acid, dinophysistoxins and azaspiracids. Three rats should be used for each test. A
diarrhetic response in any of the three rats is considered a positive result for the presence of okadaic acid, dinophysis-
toxins and azaspiracids at levels above those mentioned in Article 2 and 4.

Alternative detection methods

A series of methods such as high performance liquid chromatography (HPLC) with fluorimetric detection,
liquid
chromatography (LC)-mass spectrometry (MS), inmunoassays and functional assays such as the phosphatase inhibition
assay can be used as alternative or complementary methods to the biological testing methods, provided that either alone
or combined they can detect at least the following analogues, that they are not less effective than the biological methods
and that their implementation provides an equivalent level of public health protection:
— okadaic acid and dinophysistoxins: an hydrolysis step may be required in order to detect the presence of DTX3,
— pectenotoxins: PTX1 and PTX2,
— yessotoxins: YTX, 45 OH YTX, homo YTX, and 45 OH homo YTX,
— azaspiracids: AZA1, AZA2 and AZA3.

If new analogues of public health significance are discovered they should be included in the analysis. Standards will have
to be available before chemical analysis will be possible. Total toxicity will be calculated using conversion factors based on
the toxicity data available for each toxin.

The performance characteristics of these methods should be defined after validation following an internationally agreed
protocol.